This invention relates to an enzyme electrode and, more particularly to an enzyme electrode which is utilized for electrochemically measuring concentrations of various substrates which are to be affected by specific catalytic reactions caused by enzymes.
Furthermore, the electrode of the present invention is utilized to convert the chemical energy associated with the substrates mentioned above into electrical energy when assembled into the electrochemical cell with a counter electrode of such as an oxygen electrode.
As is well known, the energy conversion phenomena taking place inside a living organism are mainly controlled by the contributions caused by oxidoreductase, and recently, the phenomena or alternatively, the enzymatic reactions of this kind have been attempted to be utilized in the technological field.
A number of arts related to the enzyme electrode, which can be utilized for measuring the concentration of the substrate in combination with the electrochemical measuring system, have been already proposed.
Before the detailed description of the present invention proceeds, some specific aspects concerning the relation between the oxidation and reduction reaction to be catalyzed by oxidoreductase and the electrochemical measuring system are described in the following.
Generally, without the presence of an electron mediator, the oxidation and reduction reaction of the substrate catalyzed by oxidoreductase can not take place.
When the oxidation of glucose, for example, is to be taken place in the presence of glucose oxidase (GOX), oxygen O.sub.2 takes a part of the electron mediator, more particularly functioning as an electron acceptor as shown by the following reaction formula (1). EQU Glucose+O.sub.2 .sup.GOX Gluconolactone+H.sub.2 O.sub.2 ( 1)
More specifically, glucose is oxidized (dehydrogenated) to gluconolactone through the catalytic reaction caused by GOX mentioned above, while, at the same time, oxygen O.sub.2 which accepts electrons departed from glucose (electrons associated with hydrogen) is reduced to H.sub.2 O.sub.2.
In connection with the chemical process mentioned above, the respective concentrations of O.sub.2 as well as H.sub.2 O.sub.2 can be measured with the conventional electrochemical measuring method, thereby it being possible to obtain the concentration of glucose indirectly by means of the direct electrochemical measurements of O.sub.2 consumption as well as H.sub.2 O.sub.2 formation governed by the reaction formula (1) mentioned above.
As far as GOX is employed as an enzyme for the enzymatic reaction, instead of the natural electron acceptor O.sub.2 mentioned above, one of the following artificial electron acceptors such as p-benzoquinone, methyleneblue, 2,6-dichloropholindophenol, potassium ferricyanide etc., which are often called by the name of redox compound, can be applied.
More specifically, when p-benzoquinone, for example, is introduced in place of O.sub.2 in the chemical process governed by the reaction formula (1) mentioned in the foregoing, a reaction governed by the following reaction formula (2) is to take place. EQU Glucose+p-benzoquinone .sup.GOX Gluconolactone+Hydroquinone (2)
In connection with the chemical process mentioned above, since p-benzoquinone as well as hydroquinone are both electrochemically active materials, the respective concentrations of these can be directly measured and thereby, the concentration of glucose can be also indirectly measured with the governing chemical formula.
Furthermore, in addition to GOX, such oxidoreductases as xanthine oxidase, amino acid oxidase, and aldehyde oxidase etc. can accomplish the oxidation and reduction reaction concerning the substrate with one of the artificial electron acceptors defined as the redox compound mentioned in the foregoing in place of the natural electron acceptor such as O.sub.2 through the catalytic reaction thereof.
However, depending upon the nature of the oxidoreductases, some oxidoreductases need the presence of one of the compound-group termed by a coenzyme as the electron mediator to accomplish the oxidation and reduction reaction of the substrate concerned.
For example, such oxidoreductase as alcohol dehydrogenase or lactate dehydrogenase needs nicotinamide adenine dinucleotide (NAD) as the coenzyme for the respective reactions.
In addition to NAD mentioned above, nicotinamide adenine dinucleotide phosphate as well as lipoic acid are both known as the coenzyme, while NAD is a typical coenzyme.
In the circumstances where the enzymatic reaction concerning oxidation or reduction of substrates needs a coenzyme as the electron mediator, it is not practically possible to replace the coenzyme with one of the artificial redox compounds.
However, if the coenzyme is involved in the catalytic reaction in the presence of the redox compound, the substrate concentration of the chemical system becomes measurable through the electro-chemical measurement of the redox compound.
For example, when lactate dehydrogenase, NAD, and phenazine methosulfate are employed as the enzyme, the coenzyme, and the redox compound respectively, lactic acid undergoes dehydrogenation as shown below. ##EQU1##
In the chemical process governed by the chemical formula (3) mentioned above, phenazine methosulfate now comes to act as the electron mediator or more specifically, the electron acceptor due to the presence of NAD. Hence, the concentration of lactic acid in the chemical system mentioned above can be obtained by the direct electrochemical measurement of the resultant concentration of phenazine methosulfate of reduced form which is produced.
As may be clear from the description hereinabove, several kinds of attempts for connecting the enzymatic reaction with the electrochemical reaction system have been proposed. Within these attempts, special reference is directed to the conventional arts discussed below concerning the enzyme electrodes which are constituted by immobilizing oxidoreductase.
For example, U.S. Pat. No. 3,542,662 discloses an enzyme electrode comprising an immobilized enzyme membrane composed of a polyacrylicamid gel having GOX therein and covering over an oxygen permeable membrane constituting a polaragraphic oxygen electrode.
The measuring method associated with the electrode mentioned above specifies the method corresponding to that of the substrate concentration being obtained through the electrochemical measurements of the concentration of O.sub.2 which reacts with glucose in the presence of the GOX as a catalyst as described in the foregoing.
This arrangement, however, is not reliable to obtain a stable measurement, depending upon the fact that the measuring concentration of O.sub.2 itself is quite influenced by the dissolved oxygen concentration in the surroundings of the electrode concerned. Furthermore, the response time concerning the measurement tends to be delayed due to the fact that oxygen must diffuse through two membranes of the immobilized enzyme membrane and the oxygen permeable membrane before it reaches the electrode.
U.S. Pat. No. 3,838,033 discloses an enzyme electrode, in which the outside of the electron collector is covered by a semi-permeable membrane and yet, an enzyme (GOX) and redox compound are both contained in a space provided between the semi-permeable membrane and the electron collector. Furthermore, the redox compound mentioned above is contained in large excess while being present in a partially undissolved form.
The measuring method associated with the electrode mentioned above specifies the method corresponding to that of the substrate concentration obtained by the electrochemical measurement of the redox compound in the system as specifically described in the foregoing. Differing from the measuring situation concerning the oxygen concentration mentioned above in conjunction with the description of U.S. Pat. No. 3,542,662, the concentration of the redox compound remains constant, and thereby, the stable measurment is obtainable.
However, the semi-permeable membrane mentioned above is characterized in that although such a large molecule as the enzyme is not permeated, a small molecule like the substrate is freely permeated therethrough. Therefore, the redox compound whose size is almost equivalent to that of the substrate mentioned above, can be naturally permeated and thereby, gradually lost from the space mentioned above through said membrane.
A portion of the redox compound lost in a manner mentioned above can be supplemented to some extent by the dissolving of the redox compound having been maintained in the undissolved form. However, since the supplement of the redox compound mentioned above can be brought about within limit, the electrode life time does not depend upon the life time of the enzyme, but rather upon the diffusing behavior of the redox compound from the space mentioned above. The characteristic behavior mentioned above also leads to a disadvantage that the electrode life time is quite short.
Moreover, even if the microelectrode comprising the arrangements mentioned above is made to apply for the direct in-vivo measurements, it is not practically possible to utilize the electrode of this type in consideration of the adverse effect caused by the diffusional flow of the redox compounds into the living body.
In Bulletin of the Chem. Soc. of Japan 48(11), 3246, 1975, an electrode comprising an electron collector with which a collagen membrane including therein lactate dehydrogenase or alcohol dehydrogenase in their immobilized condition is brought into close contact, and its measuring characteristics of the substrate concentrations concerning lactic acid and ethanol are disclosed.
However, for every run of the measurement with the electrode mentioned above, the coenzyme (NAD for this case) as well as the redox compound (phenazine methosulfate for this case) which are substantially necessary for the measurements due to the reasons mentioned in the foregoing must be dissolved in the solution containing the substrate to be measured. Accordingly, every measuring run requires these expensive reagents without any possibility of reusing them. Moreover, the procedures used to prepare the predetermined fixed amount of the redox compound as well as that of the coenzyme for every run of the measurement will involve additional complexities for the measuring operation. As the matter of fact, the electrode of the above described type can not be applicable to the in-vivo concentration measurements due to the inherent defects mentioned in the foregoing.
As is clear from the description in the foregoing, the conventional enzyme electrodes comprising immobilized oxidoreductase still involves substantial defects and thereby, their applications are quite limited.